Approximately 30,000 Americans die every year by suicide, more deaths than those caused by HIV and homicides combined. Major depressive disorder (MDD) patients have the highest rates of suicidal behaviors, however only a subset ever commit suicide, making the identification of a molecular signature for suicide a major clinical challenge in this high risk population group. We and others have observed specific gene expression alterations in two stress response molecular mechanisms, metallothioneins and polyamines, in brains of MDD suicide victims, with opposite effects however in MDD patients who died of other causes. These stress-related mechanisms have not been carefully studied at the genetic, epigenetic, RNA, and protein level in a sample of subjects allowing the comparison between suicide, non- suicide depressed cases, and normal controls in post-mortem brain tissue. The central hypothesis of this proposal is that metallothioneins, and polyamines, two key molecular stress-response mechanisms, are impaired in MDD subjects that commit suicide. This study proposes to investigate, at the molecular level, the role played by polyamines, and metallothioneins in suicide in MDD patients. (Aim 1) We hypothesize specific gene and protein expression alterations in suicide and non-suicide MDDs compared with control subjects. Twenty MDD suicides, 20 non-suicide MDDs and 20 controls matched in terms of age and gender will be analyzed for a total of 60 subjects. We will investigate six brain regions relevant to suicide, and mood disorders, including the anterior cingulate cortex (ACC), amygdala (AMY), dorsolateral prefrontal cortex (DLPFC), hippocampus (HIPP), nucleus accumbens (NAcc), and orbitofrontal cortex (OFC) which form circuits that regulate emotional stimuli processing and change in mood states. (Aim 2) We hypothesize that stable gene and protein expression differences in suicide will be associated with genetic and epigenetic variation for the genes selected from Aim 1 using the same cohort of subjects from Aim 1. (Aim 3.1) We hypothesize that relevant biomarkers will be found in subjects for which we have both blood and brain tissue. We will attempt to validate the identified molecular targets in a new cohort of subjects (20 MDD suicides, 20 MDD non-suicides, 20 controls) (Aim 3.2) We hypothesize that medications and cortisol will have differential effects on serious MDD suicide attempters compared to MDD non-attempters, and normal controls. We will determine the effects of cortisol and antidepressants on polyamine, and metallothionein gene and protein expression. These immortalized cell lines, derived from patients with severe suicide attempts, patients without suicide attempts or thoughts, and matched controls (25 cell lines per group, 75 in total). Taken together, this project involves a large number of high quality brain samples, blood samples and a mechanistic cellular confirmation component to identify potential biomarkers in molecular stress response systems. These biomarkers might play a significant role in the identification of MDD patients with high risk of committing suicide and might offer promising targets for pharmacological interventions.